Article Figures & SI

Figures

fruA expression during development. (A) The map of the
9.5-kbp fragment containing fruA (S, StuI; H,
HincII; C, ClaI). (B) The promoter region of
fruA. The transcription initiation site is indicated by an arrow. The
DNA-binding site identified by footprint analysis shown in
Fig. 2B is
double-underlined. The sequences corresponding to oligonucleotide primers
a–d are underlined. (C) lacZ fusion analysis. The
promoter regions from nucleotides –185 to +270 (squares) and from
nucleotides –40 to +270 (circles) were fused to lacZ, and
β-galactosidase activity was measured during development. Triangles
represent pZKAT without the promoter.

Identification of the DNA-binding site. (A) Shown is a DNA-binding
assay. The probe contains the promoter region from nucleotides –185 to
–41. AS fractions prepared from vegetative (lane 2) and 4-, 8-, and 12-h
developmental cells (lanes 3, 4, and 5, respectively) were used for the
protein source. Note that the same number of initial cells is harvested at
each time point. Lane 1 contains no AS fraction. (B) Shown is
footprint analysis. DNA-binding reactions were performed under the same
conditions as described for A. After the binding reaction and gel
electrophoresis, complex I, complex II, and free probe were excised from the
gel and subjected to the treatment with 1,10-phenanthroline-copper. Lane 1,
free probe; lane 2, complex I; lane 3, complex II. Lanes G, A, T, and C
represent sequence ladders generated by a primer
5′-GATCCCCAGCCCCAATGGGAGTG-3′, which was labeled at the 5′
end with [γ-32P]ATP by T4 polynucleotide kinase and can
hybridize just upstream of the –35 region boxed in
Fig. 1B. (C)
Shown are the sequences of the DNA-binding site. (D) Sequence
comparison between regions a and b.

Identification of the fruA promoter-binding protein. (A)
Shown is purification of FBP from M. xanthus. After the second round
of DNA affinity column chromatography, the sample was applied to SDS/15% PAGE
and visualized by silver staining. (B) Purification of MrpC2 from
E. coli. After the second round of DNA affinity column
chromatography, the sample was applied to SDS/15% PAGE and visualized by
silver staining. (C) Shown are the sequences of the upstream region
of mrpC and N-terminal end of MrpC. The previously assigned
ribosome-binding site (nucleotides 1–5) and the initiation codon
(nucleotides 13–15) are underlined
(10). The N-terminal sequence
determined for FBP is double-underlined. The newly assigned initiation codon
and the ribosome-binding site are indicated by bold letters and
underlined.

Expression of fruA in M. xanthus DZF1 and
mrpC::km. fruA expression was examined by primer
extension analysis as described
(18). Total RNA was prepared
from DZF1 and mrpC::km during vegetative growth (0 h) and
fruiting body development (6 and 12 h). As a control, vegA expression
was examined. Oligonucleotide primers
5′-TTGACTTTCAGCTACTCCTGACG-3′ and
5′-GCTTTATCCACGGACATT-3′ were used for fruA and
vegA, respectively.

DNA-binding analysis. (Upper) Three kinds of probes (10 fmol), an
HindIII (H)–BamHI (B) fragment containing both regions
a and b (lanes 1–3), an SmaI (S)–BamHI fragment
containing region a (lanes 4–6), or an
HindIII–SmaI fragment containing region b (lanes
7–9) were used for DNA-binding reactions in a 10-μl volume. Note that
the HindIII and BamHI sites are from the cloning vector. No
protein was added for lanes 1, 4, and 7. Purified MrpC2 was added for lanes 2,
5, and 8 (1 ng) and lanes 3, 6, and 9 (2 ng). MrpC–DNA complexes are
indicated by arrowheads. (Lower) The fruA promoter region
from nucleotides –185 to –41 is shown.

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